Post-transcriptional gene silencing and modification techniques have shown great promise for the treatment of disease. Gene silencing techniques are designed to repress the expression of a gene by interfering with the processing of its mRNA transcript, while modification techniques offer the possibility to correct errors in the transcript.
Currently, the main techniques for post-transcriptional gene silencing are the use of antisense oligodeoxyribonucleic acids (ODNs) and RNA interference (RNAi). Both of these methods have been developed for use both in cell culture experiments and as therapeutics.
Antisense ODNs act by hybridizing to pre-mRNAs and mRNAs to form a substrate for ribonuclease H (RNase H). RNase H then acts to cleave the ODN-RNA duplex, destroying the RNA target and preventing its expression. ODNs that are resistant to the action of RNase H can also be used to sterically inhibit splicing of a pre-mRNA. For example, an ODN can be designed to hybridize across the exon-intron junction of a pre-mRNA, preventing that pre-mRNA from being spliced and expressed.
ODNs have shown limited success in gene silencing, and their use has several disadvantages. These disadvantages stem primarily from the fact that high concentrations of ODNs must be used to elicit effective gene silencing. Use of high concentrations of ODNs, especially those of more than 20 nucleotides in length, can trigger an immune response and the production of interferons. ODNs can also bind endogenous proteins, causing unintended toxic side effects. Further, because they are synthetic oligonucleotides, ODNs may only be delivered by exogenous means, such as injection, limiting their use as a long term therapeutic.
RNAi is able to successfully cause gene silencing at concentrations at least 100 times less than those necessary for successful silencing with ODNs. In RNAi, double stranded RNA molecules or microRNA hairpins are cleaved into 21-28 fragments, which are assembled into a RNA induced silencing complex (RISC). The RISC then causes the degradation of mRNAs that contain sequence complementary to the 21-28 nucleotide fragment. Further, RNAi effector molecules are able to be transcribed from DNA, allowing for delivery of the effector molecules by a variety of methods, such as through use of viral vectors.
Because of the above advantages, RNAi has become the primary method of post-transcription gene silencing. However, the technique has several disadvantages. The first of these disadvantages is that RNAi can only be used for targeting exons, making it ineffective against pre-mRNA transcripts. This is especially important for cell culture studies where it is desirable to transfect cells with a vector encoding an exogenous version of the protein whose expression has been knocked down. As RNAi targets only exons, it will also target this exogenous transcript.
Another disadvantage of RNAi is that certain mRNA targets are refractory to its use. Because of the nature of RNAi, it is not possible to improve targeting, meaning that some genes are simply not able to be silenced with RNAi. This has complicated its use as a therapeutic. Thus far, researchers have not had widespread success in using RNAi for silencing genes in mammals in vivo.
Because of the defects in the gene silencing techniques known in the art, it is desirable to develop improved methods of post-transcriptional gene silencing. Such methods could be used as substitutes for the above techniques. Further, new methods of gene silencing could be used in combination with known techniques, especially in cell culture experiments.
U.S. Pat. No. 5,972,705 to Fournier et al., which is hereby incorporated by reference herein, discloses a method of using small nucleolar (sno) RNAs to cause the 2′-O-methylation of specific nucleotides in an mRNA. Fournier teaches use of 2′-O-methyl (2′-OMe) modifications at the 5′ or 3′ splice sites of pre-mRNA to prevent the cleavage of the pre-mRNA that occurs during the splicing process. In certain circumstances, blocking cleavage at the 5′ or 3′ splice site may prevent pre-mRNA splicing and subsequent expression. However, as the 2′-hydroxyl group of the nucleotide at either the 5′ or 3′ splice sites is not involved in the splicing process, it is not clear that modification at either of these sites will block splicing at all.
Although Fournier presents method of pre-mRNA modification for post-transcriptional gene silencing, the method as described has a disadvantage in that it is unable to prevent the splicing and subsequent expression of many alternatively spliced transcripts. As alternatively spliced pre-mRNAs have more than one 5′ or 3′ splice site, blocking the cleavage of one site will only allow the transcript to splice with another splice site, still leading to expression of the gene. Further, there are a significant number of exons that contain alternative 5′ or 3′ splice sites just up or down stream from the main splice site (Ast, Nature Rev. Genet. 5:773, 2004). Hence, blocking a main splice site would only lead to the use of the alternative site, and would not prevent expression of the transcript. The inability to block gene expression of certain alternatively spliced transcripts is especially significant disadvantage for human therapeutic applications, as 50% of human transcripts are estimated to be alternatively spliced.
Post transcriptional mRNA modification also has the potential to correct errors in pre-mRNA or mRNA transcripts. Modification of an mRNA transcript may cause a mutated codon to be read differently, allowing for the correction of a mutation on an mRNA level. Correction on the mRNA level may be desirable over correction at the DNA level, as genetic therapy techniques have been used with limited success and at excessive cost. Further, there is likely to be less risk of unintended consequences (such as causing a mutation elsewhere) involved in modification of the temporal mRNA transcript as compared to modification of a DNA encoded gene.
Nonsense mutations are one of the main types of mutations that may lend themselves to correction on the mRNA transcript level. A nonsense mutation is a mutation that creates an early stop codon in the coding sequence. As an early stop codon (or nonsense codon) is created, the translation machinery stops before the entire coding sequence is read, and a truncated version of the protein being encoded is formed. The truncated forms of these proteins may lead to disease pathologies such a Cystic Fibrosis and Duchene Muscular Dystrophy, among other diseases.
US Published Patent Application No. 2006/0035943 to Karp, which is hereby incorporated herein, describes use of a chemical compound to cause the translation process to bypass nonsense codons. While this method may cause the translation machinery to bypass the desired nonsense codon, it can also cause other, legitimate stop codons to be bypassed, potentially leading to undesired consequences.
Overall, there is a need in the art for methods for modifying both pre-mRNA and mRNA transcripts to prevent or modify the expression of the protein coded in those transcripts. As these methods would provide for specific targeting of sites on mRNAs, they would overcome many of the drawbacks and risks of RNA interference and gene therapy techniques.